Alveolar rhabdomyosarcoma is an aggressive childhood muscle cancer associated with significant morbidity and mortality. Eighty-five percent of alveolar rhabdomyosarcomas demonstrate a common genetic alteration, the translocation-mediated fusion of a developmentally regulated Pax transcription factor (either Pax3 or Pax7) to the Forkhead transcription factor, Fkhr. An understanding of the molecular events underlying the development of alveolar rhabdomyosarcomas will improve our ability to stratify patients to risk-based therapies and to develop new therapies. The goals of this project are to investigate whether the postembryonic, translocation-mediated fusion of Pax3 to Fkhr initiates an aberrant embryonic muscle development program which is involved in the genesis of alveolar rhabdomyosarcoma, and to investigate whether the early and intermediate steps in the activation of oncogenic properties contributing to the genesis of alveolar rhabdomyosarcoma occur in a predictable sequence. The specific aims of this proposal are (I) to examine whether a genomic single copy-number of Pax3:Fkhr is sufficient and necessary to cause both aberrant Pax3 pathway activation and the initiation of alveolar rhabdomyosarcoma, (II) to study whether genetic, transcriptional, or translational contributions to common oncogenic mechanisms (eg. insensitivity to antigrowth signals, evasion of apoptosis) occur in a predictable sequence in alveolar rhabdomyosarcomas, and (III) to determine whether Cre recombinase-mediated translocations between heterologous chromosomes can be induced somatically at a frequency sufficient for the initiation of alveolar rhabdomyosarcoma. For the first and second aims, a conditional, knock-in mouse model of alveolar rhabdomyosarcoma will be created which initially harbors two functionally normal Pax3 alleles. Conditional, somatic expression of the site-specific DNA recombinase Cre will mediate a genomic rearrangement converting a single Pax3 allele to a single Pax3:Fkhr allele in skeletal muscle only. Animals will be serially sacrificed to analyze the ensuing sequence of intracellular changes in cells harboring Pax3:Fkhr. Analysis will be performed at the genomic, mRNA, and protein level with an emphasis on known markers of tumor progression. For the final aim, a conditional, somatic translocation between the Pax3 and Fkhr loci will be attempted using Cre-mediated rearrangement of heterologous chromosomes. Dr. Keller's research training with Dr. Capecchi and the support of the NCI will prepare Dr. Keller for an independent research career investigating developmental aspects of pediatric cancer. Ultimately, a detailed knowledge of dysregulated developmental pathways in pediatric malignancies may improve our ability to treat childhood cancer.